Tetracycline process



United States Patent TETRACYCLINE PROCESS Fred W. Tanner, Jr., Baldwin, N.Y., assignor to Chas. Pfizer &'Co. Inc., New York, N.Y., a corporation of Delaware No Drawing. Filed Mar. 11, 1957, Ser. No. 645,024 5 Claims. c1. 19s s0 This application is concerned with a new and useful process for the production of tetracycline. More particularly, it is concerned with a'process by which tetracycline is preferentially produced by the addition of a chemical agent to a fermentation medium which in the absence of this agent produces a major proportion of or even only chlortetracycline.

Tetracycline is a broad spectrum antibiotic the therapeutic efiicacy of which has been attested by a large number of medical investigators as reported in many articles in the medical literature. Its chemical structure was first reported in the Journal of the American Chemical Society, volume 74, page 4976 (1952). Its chemical relationship to chlortetracycline and oxytetracycline is Well known.

Tetracycline is generally produced by one of two methods. These methods are catalytic dehalogenation of chlortetracycline with hydrogen, and fermentation. In both of these methods, there are definite disadvantages. The catalytic dehalogenation of chlortetracycline requires that the antibiotic be isolated from a fermentation source in relatively high purity and then subjected to chemical action requiring expensive processing equipment and catalysts. The fermentation method, although economically more practical, produces not only tetracycline but also chlortetracycline. In most media the latter is generally produced preferentially. This necessitates expensive purification procedures to separate the two antibiotics.

Because of its high activity and because of its high stability in certain media, tetracycline is preferred in many applications over other. antibiotics. It is apparent, therefore, that any process which makes it possible to produce tetracycline substantially uncontaminated by chlortetracycline or Without the necessity of chemical processing fulfills a definite and long-felt need in the art.

The usual tetracycline-producing microorganisms. belong to the genus Streptomyces. The particular strains which are'usually used are Streptomyces aureofaciens, for example, Streptomyces aureofacz'ens (NRRL 2209), and Streptomyces viridifaciens, for example, Streptomyces viridifaciens' {-ATCC 11989). These microorganisms produce fermentation products containing substantially only chlortetracycline. Many attempts have been made to alter the usual fermentation media in order to sup press 'chlortetacycline in favor of tetracycline in the produced antibiotic mixture. Two of these are described in the patent literature. US. Patent 2,739,924 issued to Lein and Gourevitch describes the addition of a bromide and the like, for example, sodium bromide to fermentation media. U.S. Patent 2,734,018 issued to Minie'ri et a1. describes the use of a dechlo'rinated fermentation media. While both of the described processes are capable of producing broths of increased tetracycline content, neither of them is completely satisfactory in that the amount of chlortetracycline produced is still relatively high. Furthermore, the dechlorination procedure is laborious and expensive.

Patented June 14,1960

It has now most unexpectedly been discovered that the addition of Z-benzoxazolyl thiol N-methyl carbamate surprisingly makes it possible to produce tetracycline and chlortetracycline in mixtures containing as high as 98% tetracycline or even higher. The 2-benzoxazolyl thiol N-rnethyl carbamate may be substituted on the carbon atoms of the phenyl ring with halogen atoms or with such groups as lower alkyl, hydroxyl, nitro, mercapto and others, but the unsubstituted compound is highly active per se. Other variations in the structure of the basic molecule may be made without seriously interfering with the desirable activity.

In practicing the instant invention a 2-benzoxazolyl thiol N-methyl carbamate is added to a fermentation broth of a tetracycline-producing microorganism of the genus Streptomyces and aerobic fermentation is allowed to continue in the normal way.

The fermentation may be carried out in accordance with either the well known submerged or surface techniques. When the antibiotic activity of the fermentation broth is isolated, for example, by extraction and the antibiotic content analyzed, it is found that the amount of tetracycline produced is substantially higher than is produced in the absence of Z-benzoxazolyl thiol N-methyl carbamate. So surprisingly efncient is the process of this invention that concentrations of the tetracyclineproducing agent as low as twenty parts per million produce broths containing up to 200 ug/ml. of tetracycline of greater than purity. The activity and purity of the tetracycline produced were established by chromatographic methods in which known solutions of tetracycline and chlortetracycline and mixtures of these 'wer used as reference standards.

Control experiments run under exactly the same conditions and evaluated in exactly the same manner produced broths containing 200 ,ug./ml. 'of chloretracycline and only a trace of tetracycline. Similarly high ratios of tetracycline to chlortetracycline are achieved With concentrations of 2-benzoxazolyl thiol N-methyl carbamate as low as 0.2 part per million or even lower. With most chlortetracycline producing microorganisms concentrations of about twenty partsper million are entirely adequate but the optimum concentration may vary somewhat with the particular strain or mutant of microorganism being used. Thus certain microorganisms may yield optimum amounts of tetracycline at concentrations higher or lower than twenty parts" per million. It is conceivable that with some particular strains or mutants the toxicity level may be higher or lower than. with the microorganisms heretofore tested. The optimum concentration may depend in some cases on whether the 2-benzoxazolyl thiol N-methyl carbamate is added before or after inoculation of the fermentation media. For most applications, however, concentrations as high as 100 parts per million or even higher are tolerable. It is to be noted that the desirable effect of obtaining extremely high tetracycline to chlortetracycline ratio in the produced antibiotic is achieved without the necessity of expensive dechlorination procedures being applied to the fermentation media.

Many suitable fermentation media of the type generally used in the production of antibiotics by fermentation methods can be used in practicing this invention. These media will usually contain a source of carbohydrates and a source of nitrogen preferably in organic form. A variety of different metallic salts also have some value in, stimulating production of the antibiotic. Among the carbohydrate solutions which are useful are molasses, glucose, starches, glycerol, etc. Organic nitrogen sources include soybean meal, Wheat gluten, peanut meal, hydrolyzate of "casein and other proteins. Certain crude materials contain growth stimulatory substances which are of some value in obtaining maximum or scope thereof.

a 300 ml. Erlenmeyer flask.

V j yields of the antibiotic. These include such substances" as corn steep liquor, distillers solubles, yeast extract,vetc.

' Salts such as sodium nitrate, sodium chloride, potassium Soybean meal fi 40 Sodium nitrate 3 2-benzoxazolyl thiol N-methyl carbamate 85 100 ml. of tap water.

dition of oils such as lard oil, soybean oil, etc. may be advisable. Useful media are described in US. Patents 2,482,055, 2,709,672 and 2,776,243 as well as in now abandoned US. patent applications, Serial Nos. 3923, 0,

, 391,709 and 391,710, filed November 12, 1953.

In practicing the instant invention the active agent may be added to the fermentation media before or after in oculation. .It is, however, preferably added before significant amounts of antibiotic activity have been produced since prior to the addition of Z-benzoxazolyl thiol N-methyl carbamate the antibiotic produced is preponderantly chlortetracycline. i

The following examples are given solely for the purpose of illustration only and are not to be construed as limitations of this invention, many apparent variations of which are possible without departing from the spirit Example I A spore suspension of Streptomyces aureofaciens was introduced into 100 ml. of the following composition in Grams per liter Sucrose Corn steep liquor 10 Ammonium monohydrogen sulfate 2 Calcium carbonate 2 After forty-eight hours incubation at 28 C. on a rotary shaker, approximately 2.5 ml. of this culture served to inoculate'one liter of a fermentation medium of the following composition.

Cornstarch g. per liter.

2-benzoxazolyl thio N -methyl carbamate 20 mg./l.

The medium was autoclaved forty'minutes at twenty pounds steam pressure. After approximately ninety hours incubation at 28 C. on a rotary shaker, samples of the broth were withdrawn and tested by paper chromatographic methods for antibiotic content. It was found that the broth contained 200 ,ugJml. of tetracycline and that this represented more than 98% of the total antibiotic activity produced. The remainder of the antibiotic activity was attributed to chlortetracycline.

. Example II The procedure of Example I was repeated except that the Z-benzoxazolyl thiol N-methyl carbamate was added twenty-four hours after incubation and the incubation was continued for an additional sixty-six hours at 28 C. Analysis of aliquot portions of the broth established a tetracycline content of 200 ugJ-ml. of about 95% purity.

Example III broth established a tetracycline content of 200 ,ug./ ml. of

of about 97% purity.

. Example IV A spore suspension of Streptomyces aureofaciens (NRRL 2209) prepared as described in Ex'ampleI was The medium was autoclaved forty minutes at twenty pounds steam pressure. After 100 hours incubation at 35 C. in a rotary shaker, samples were withdrawnand tested for antibiotic activity. It was foundlthat the broth contained 200 rig/ml. of tetracycline and that this represented more than 98% of the total antibiotic, activity pro duced.

7 E p e V A spore suspension of Streptomyces viridifaciens prepared as in Example I'was used to inoculate 100 ml. of a sterile nutrient medium having the following composition. 7 a a Soybean meal 40 g. per liter. Sodium nitrate 3 g. per liter. Z-benzoxazolyl thiol N-rnethyl carbamate. 2 mg. per liter. 100 ml. of tap water. v V The medium was autoclaved forty minutes at twenty pounds steam pressure. After 100 hours incubation at 35 C. in a rotary shaker, samples were withdrawn and tested for antibiotic activity. It was found that the broth contained 200 gJml. of tetracycline and that this represented more than of the total antibiotic activity produced.

What is claimed is:

1. In a process for producing tetracycline by aerobic fermentation with a chlortetracycline-producing microorganism of the genus Streptomyces, the improvement which comprises conducting the fermentation in the presence of 2-benzoxazolyl thio N-methyl carbamate.

2. In a process for producing tetracycline'by aerobic fermentation with a chlortetracycline-producing microorganism of the species Strptomyces aureofaciens, the improvement which comprises conducting the fermentatron 1n the presence of Z-benzoxazolyl thiol -N-'methyl carbamate. e

3. In a process for producing-tetracycline by aerobic fermentation with a chlortetracycline-producing microorganism of the species Stz'eptomyces viridifaciens, the lmprovement which comprises conducting the fermentatron 1n the presence of 2-benzoxazolyl thiol N-methyl carbamate. 7 e a 4. A'process as in claim 1 wherein -from 0.2.to parts per million of 2-benzoxazolyl thiol N-methyl carbamate is used. V

5. In a process of aerobic fermentation of a nutrient medium with a microorganism of the genus Streptomyces, said fermentation normally producing an antibiotic consistmg substantially of chlort'etracycline with minor amounts of tetracycline, the improvement com-prising suppressing the production of chlortetracycline in favor of tetracycline by the addition of 2-benzoxazolyl thiol N- methyl carbamate to the fermentation medium. 7

References Cited in the file of this patent 'UNITED STATES PATENTS 2,712,517 Gourevith et al. July 5, 1955 2,734,018 Minieri et al. Feb. 7, 1956 2,804,476 Bogert'et a1. Aug. 27, 1957 V FOREIGN PATENTS 316,291 Switzerland -Q Nov. 15, 1956 

1. IN A PROCESS FOR PRODUCING TETRACYCLINE BY AEROBIC FERMENTATION WITH A CHLORTETRACYCLINE-PRODUCING MICROORGANISM OF THE GENUS STREPTOMYCES, THE IMPROVEMENT WHICH COMPRISES CONDUCTING THE FERMENTATION IN THE PRESENCE OF 2-BENZOXAZOLYL THIO N-METHYL CARBAMATE. 